Mesoscale polynucleotide amplification device and method

ABSTRACT

Disclosed are devices for amplifying a preselected polynucleotide in a sample by conducting a polynucleotide polymerization reaction. The devices comprise a substrate microfabricated to define a sample inlet port and a mesoscale flow system, which extends from the inlet port. The mesoscale flow system includes a polynucleotide polymerization reaction chamber in fluid communication with the inlet port which is provided with reagents required for polymerization and amplification of a preselected polynucleotide. In one embodiment the devices may be utilized to implement a polymerase chain reaction (PCR) in the reaction chamber (PCR chamber). The PCR chamber is provided with the sample polynucleotide, polymerase, nucleoside triphosphates, primers and other reagents required for the polymerase chain reaction, and the device is provided with means for thermally controlling the temperature of the contents of the reaction chamber at a temperature controlled to dehybridize double stranded polynucleotide, to anneal the primers, and to polymerize and amplify the polynucleotide.

This is a continuation of application Ser. No. 07/877,662 filed on May1, 1992, now abandoned.

REFERENCE TO RELATED APPLICATIONS

Ths application is being filed contemporaneously with the followingrelated applications: U.S. Ser. No. 07/877,702, filed May 1, 1992,abandoned; U.S. Ser. No. 07/877,701, filed May 1, 1992, abandoned; U.S.Ser. No. 07/877,536 filed May 1, 1992, now U.S. Pat. No. 5,304,487; andU.S. Ser. No. 07/877,661, filed May 1, 1992, now U.S. Pat. No.5,296,375; the disclosures of which are incorporated herein byreference.

BACKGROUND OF THE INVENTION

This invention relates generally to methods and apparatus for conductinganalyses. More particularly, the invention relates to the design andconstruction of small, typically single-use, modules capable of analysesinvolving polymerase chain reaction (PCR).

In recent decades the art has developed a very large number ofprotocols, test kits, and cartridges for conducting analyses onbiological samples for various diagnostic and monitoring purposes.Immunoassays, agglutination assays, and analyses based on polymerasechain reaction, various ligand-receptor interactions, and differentialmigration of species in a complex sample all have been used to determinethe presence or concentration of various biological compounds orcontaminants, or the presence of particular cell types.

Recently, small, disposable devices have been developed for handlingbiological samples and for conducting certain clinical tests. Shoji etal. reported the use of a miniature blood gas analyzer fabricated on asilicon wafer. Shoji et al., Sensors and Actuators, 15:101-107 (1988).Sato et al. reported a cell fusion technique using micromechanicalsilicon devices. Sato et al., Sensors and Actuators, A21-A23:948-953(1990). Ciba Corning Diagnostics Corp. (USA) has manufactured amicroprocessor-controlled laser photometer for detecting blood clotting.

Micromachining technology originated in the microelectronics industry.Angell et al., Scientific American, 248:44-55 (1983). Micromachiningtechnology has enabled the manufacture of microengineered devices havingstructural elements with minimal dimensions ranging from tens of microns(the dimensions of biological cells) to nanometers (the dimensions ofsome biological macromolecules). This scale is referred to herein as"mesoscale". Most experiments involving mesoscale structures haveinvolved studies of micromechanics, i.e., mechanical motion and flowproperties. The potential capability of mesoscale structures has notbeen exploited fully in the life sciences.

Brunette (Exper. Cell Res., 167:203-217 (1986) and 164:11-26 (1986))studied the behavior of fibroblasts and epithelial cells in grooves insilicon, titanium-coated polymers and the like. McCartney et al. (CancerRes., 41:3046-3051 (1981)) examined the behavior of tumor cells ingrooved plastic substrates. LaCelle (Blood Cells, 12:179-189 (1986))studied leukocyte and erythrocyte flow in microcapillaries to gaininsight into microcirculation. Hung and Weissman reported a study offluid dynamics in micromachined channels, but did not produce dataassociated with an analytic device. Hung et al., Med. and Biol.Engineering, 9:237-245 (1971); and Weissman et al., Am. Inst. Chem. Eng.J., 17:25-30 (1971). Columbus et al. utilized a sandwich composed of twoorthogonally orientated v-grooved embossed sheets in the control ofcapillary flow of biological fluids to discrete ion-selective electrodesin an experimental multi-channel test device. Columbus et al., Clin.Chem., 33:1531-1537 (1987). Masuda et al. and Washizu et al. havereported the use of a fluid flow chamber for the manipulation of cells(e.g. cell fusion). Masuda et al., Proceedings IEEE/IAS Meeting, pp.1549-1553 (1987); and Washizu et al., Proceedings IEEE/IAS Meeting pp.1735-1740 (1988). The art has not fully explored the potential of usingmesoscale devices for the analyses of biological fluids.

Methodologies for using polymerase chain reaction (PCR) to amplify asegment of DNA are well established. (See e.g., Maniatis et al.Molecular Cloning: A Laboratory Manual, Cold Spring Harbor LaboratoryPress, 1989, pp. 14.1-14.35.) A PCR amplification reaction can beperformed on a DNA template using a thermostable DNA polymerase, e.g.,Taq DNA polymerase (Chien et al. J. Bacteriol.:127:1550 (1976)),nucleoside triphosphates, and two oligonucleotides with differentsequences, complementary to sequences that lie on opposite strands ofthe template DNA and which flank the segment of DNA that is to beamplified ("primers"). The reaction components are cycled between ahigher temperature (e.g., 94° C.) for dehybridizing ("melting") doublestranded template DNA, followed by a lower temperature (e.g., 65° C.)for annealing and polymerization. A continual reaction cycle betweendehybridization, annealing and polymerization temperatures providesexponential amplification of the template DNA. For example, up to 1 μgof target DNA up to 2 kb in length can be obtained from 30-35 cycles ofamplification with only 10⁻⁶ μg of starting DNA. Machines for performingautomated PCR chain reactions using a thermal cycler are available(Perkin Elmer Corp.)

PCR amplification has been applied to the diagnosis of genetic disorders(Engelke et al., Proc. Natl. Acad. Sci., 85:544 (1988), the detection ofnucleic acid sequences of pathogenic organisms in clinical samples (Ouet al., Science, 239:295 (1988)), the genetic identification of forensicsamples, e.g., sperm (Li et al., Nature, 335:414 (1988)), the analysisof mutations in activated oncogenes (Farr et al., Proc. Natl. Acad.Sci., 85:1629 (1988)) and in many aspects of molecular cloning (Oste,BioTechniques, 6:162 (1988)). PCR assays can be used in a wide range ofapplications such as the generation of specific sequences of cloneddouble-stranded DNA for use as probes, the generation of probes specificfor uncloned genes by selective amplification of particular segments ofcDNA, the generation of libraries of cDNA from small amounts of mRNA,the generation of large amounts of DNA for sequencing, and the analysisof mutations. There is a need for convenient rapid systems for PCRanalyses, which could be used clinically in a wide range of potentialapplications in clinical tests such as tests for paternity, and geneticand infectious diseases.

An object of the invention is to provide analytical systems with optimalreaction environments that can analyze microvolumes of sample, detectvery low concentrations of a polynucleotide, and produce analyticalresults rapidly. Another object is to provide easily mass produced,disposable, small (e.g., less than 1 cc in volume) devices havingmesoscale functional elements capable of rapid, automated PCR analysesof a preselected cell or cell-free sample, in a range of applications.It is a further object of the invention to provide a family of suchdevices that individually can be used to implement a range of rapidclinical tests, e.g., tests for viral or bacterial infection, tests forcell culture contaminants, or tests for the presence of recombinant DNAor a gene in a cell, and the like.

SUMMARY OF THE INVENTION

The invention provides a family of small, mass produced, typicallyone-use devices for conducting a polynucleotide polymerization reactionto enable the rapid amplification of a polynucleotide in a sample. Inone embodiment, the device comprises a solid substrate, on the order ofa few millimeters thick and approximately 0.2 to 2.0 centimeters square,that is microfabricated to define a sample inlet port and a mesoscaleflow system. The flow system of the device includes a sample flowchannel extending from the inlet port, and a polynucleotidepolymerization reaction chamber in fluid communication with the flowchannel polynucleotide. The term "mesoscale" is used herein to definechambers and flow passages having a cross-sectional dimension on theorder of 0.1 μm to 500 μm, with preferred reaction chamber widths on theorder of 2.0 to 500 μm, more preferably 3-100 μm. For many applications,channels of 5-50 μm widths will be useful. Chambers in the substratewherein amplification takes place may have somewhat larger dimensions,e.g., 1-5 mm. Preferred reaction chamber and channel depths are on theorder of 0.1 to 100 μm, typically 2-50 μm. The flow channels in thedevices, leading to the reaction chambers, have preferred widths on theorder of 2.0 to 200 μm and depths on the order of 0.1 to 100 μm.

In one embodiment, the devices may be utilized to implement apolymerization chain reaction (PCR) in the reaction chamber. Thereaction chamber may be provided with reagents for PCR including asample polynucleotide, polymerase, nucleoside triphosphates, a firstprimer hybridizable with the sample polynucleotide, and a second primerhybridizable with a sequence that is complementary to the samplepolynucleotide, wherein the first and second primers define the terminiof the polymerized polynucleotide product. The device also may includemeans for thermally cycling the contents of the PCR chamber, such that,in each cycle, the temperature is controlled to 1) dehybridize ("melt")double stranded polynucleotide, 2) anneal the primers to single strandedpolynucleotide, and 3) synthesize amplified polynucleotide between theprimers. In one embodiment, the PCR chamber may comprise one sectionwhich is thermally cycled sequentially to the required temperatures forPCR. Alternatively, the PCR chamber may comprise two or more sections,set at the different temperatures required for dehybridization,annealing and polymerization, in which case the device further comprisesmeans for cycling the contents of the chamber between the sections toimplement the PCR, e.g., a pump or other means as disclosed herein. Thedevice may further include means for detecting the amplifiedpolynucleotide. The devices may be used to implement a variety ofautomated, sensitive and rapid polynucletide analyses, includinganalyses for the presence of polynucleotides in cells or in solution, orfor analyses for a virus or cell types using the presence of aparticular polynucleotide as a marker.

Generally, as disclosed herein, the solid substrate comprises a chip,containing the mesoscale flow system and the reaction chamber(s). Themesoscale flow channels and reaction chambers may be designed andfabricated from silicon and other solid substrates using establishedmicromachining methods. The mesoscale flow systems in the devices may beconstructed by microfabricating flow channels and one or more reactionchambers into the surface of the substrate, and then adhering a cover,e.g., a transparent glass cover, over the surface. The devices analyzemicrovolumes (<10 μL) of a sample, introduced into the flow systemthrough an inlet port defined, e.g., by a hole communicating through thesubstrate or the cover. The volume of the mesoscale flow systemtypically will be <5 μL, and the volume of individual channels,chambers, or other functional elements are often less than 1 μL, e.g.,in the nanoliter or even picoliter range. Polynucleotides present invery low concentrations, (e.g. nanogram quantities) can be rapidlyamplified (<10 minutes) and detected. After a polynucleotidepolymerization assay is complete, the devices may be discarded.

The chips typically will be used with an appliance which contains anesting site for holding the chip, and which mates one or more inputports on the chip with one or more flow lines in the appliance. After abiological fluid sample suspected to contain a particular polynucleotideis applied to the inlet port of the substrate, the chip is placed in theappliance and a pump, e.g., in the appliance, is actuated to force thesample through the flow system. Alternatively, a sample may be injectedinto the chip by the appliance. Reagents required for the assay, such asa polymerase, may be added to the polynucleotide sample prior toinjection into the chip. Alternatively, reagents necessary to completethe assay can be injected into the reaction chamber from a separateinlet port, e.g., by the appliance. Fluid samples and reagents may alsoenter the mesoscale flow system by capillary action.

In one embodiment, the devices may be utilized to perform a PCR assay,and the temperature of one or more section(s) in the reaction chambercan be regulated by, e.g., providing one or more electrical resistanceheaters in the substrate near the reaction chamber, or by using a pulsedlaser or other source of electromagnetic energy directed to the reactionchamber. The appliance may include electrical contacts in the nestingregion which mate with contacts integrated into the structure of thechip, e.g., to power electrical resistance heating of the reactionchamber. A cooling element may also be provided in the appliance toassist in the thermal regulation of the reaction chamber. The appliancemay be provided with conventional circuitry sensors in communicationwith sensors in the device for thermally regulating the PCR temperaturecycles required for the dehybridization and polymerization reactions.

The amplified polynucleotide produced by the polynucleotideamplification reaction in the mesoscale reaction chamber can becollected through a port in the substrate and detected, e.g., by gelelectrophoresis or any other method. Alternatively, a mesoscaledetection region may be microfabricated in the substrate, in fluidcommunication with the reaction chamber in the device, as a part of themesoscale flow system. The detection region may include a labeledbinding moiety, such as a labeled polynucleotide or antibody probe,capable of detectably binding with the amplified polynucleotide. Thepresence of polymerized polynucleotide product in the detection regioncan be detected, e.g., by optical detection of agglutination of thepolymerized polynucleotide and the binding moiety through a glass coverover the detection region or through a translucent section of thesubstrate itself.

A positive assay may also be indicated by detectable changes in samplefluid flow properties such as changes in pressure or electricalconductivity at different points in the flow system upon production ofpolymerized polynucleotide in the reaction chamber. In one embodiment,the device comprises a mesoscale flow system which includes apolynucleotide amplification reaction chamber, and a detection region isused in combination with an appliance which includes sensing equipmentsuch as a spectrophotometer capable of reading a positive result throughan optical window, e.g., disposed over the detection region. Theappliance may also be designed to receive electrical signals indicativeof a pressure reading, conductivity, or the like, sensed in the reactionchamber, the detection region, or some other region of the flow system.

The substrate may comprise a plurality of detection/reaction chambers toenable the rapid parallel detection of polynucleotides in a mixture. Themesoscale flow system may include protrusions, or a section of reducedcross sectional area, to enable the lysis of cells in the microsampleprior to delivery to the reaction chamber. Sharp edged pieces ofsilicon, trapped in the flow path, can also be used as a lysis means.The mesoscale flow system also may include a cell capture regioncomprising a binding moiety, e.g., immobilized on a wall of a flowchannel, which binds a particular type of cell in a heterogeneous cellpopulation at a low fluid flow rate, and at a greater flow rate,releases the cell type prior to delivery of the cells to a cell lysisregion then to a reaction chamber. In this embodiment, intracellular DNAor RNA is isolated from a selected cell subpopulation and delivered tothe mesoscale reaction chamber for polynucleotide analysis in onedevice.

In another embodiment, magnetic beads may be provided within themesoscale flow system, which can be moved along the flow system by anexternal magnetic field, e.g., in the appliance. In one embodiment, apolynucleotide probe may be immobilized on the magnetic beads enablingthe beads to bind to amplified polynucleotide in the reaction chamber.Magnetic beads containing an immobilized polynucleotide probe may be,e.g., transported through the flow system to the reaction chamber at theend of an assay to bind to the polymerized polynucleotide product. Thebound polynucleotide may then be transported on the magnetic beads to adetection or purification chamber in the flow system, or to a collectionport.

Some of the features and benefits of the devices are illustrated inTable 1. The devices can provide a rapid test for the detection ofpathogenic bacteria or viruses, or for the presence of certain celltypes, or the presence of a gene or a recombinant DNA sequence in acell. The devices as disclosed herein are all characterized by amesoscale flow system including a PCR chamber which is used to amplify apolynucleotide in a sample, which may be provided with polymerase andother reagents required for PCR. The device may be used to amplify apolynucleotide in a wide range of applications. At the conclusion of theassay the chip typically is discarded.

                  TABLE 1                                                         ______________________________________                                        Feature       Benefit                                                         ______________________________________                                        Flexibility   No limits to the number of chip                                               designs or applications available.                              Reproducible  Allows reliable, standardized, mass                                           production of chips.                                            Low Cost      Allows competitive pricing with                                 Production    existing systems. Disposable nature                                           for single-use processes.                                       Small Size    No bulky instrumentation required.                                            Lends itself to portable units and                                            systems designed for use in non-                                              conventional lab environments.                                                Minimal storage and shipping costs.                             Microscale    Minimal sample and reagent volumes                                            required. Reduces reagent costs,                                              especially for more expensive,                                                specialized test procedures. Allows                                           simplified instrumentation schemes.                             Sterility     Chips can be sterilized for use in                                            microbiological assays and other                                              procedures requiring clean                                                    environments.                                                   Sealed System Minimizes biohazards. Ensures                                                 process integrity.                                              Multiple Circuit                                                                            Can perform multiple processes or                               Capabilities  analyses on a single chip. Allows                                             panel assays.                                                   Multiple      Expands capabilities for assay and                              Detector      process monitoring to virtually any                             Capabilities  system. Allows broad range of                                                 applications.                                                   Reuseable Chips                                                                             Reduces per process cost to the user                                          for certain applications.                                       ______________________________________                                    

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic longitudinal cross sectional view of a deviceaccording to the invention that includes a solid substrate 14, on whichis machined mesoscale flow channel 20 connected to inlet ports 16 andPCR reaction chamber 22, with a transparent cover 12 adhered to thesurface of the substrate.

FIG. 2 is a perspective view of the device of FIG. 1.

FIG. 3A is a schematic illustration of an analytical device 10 nestedwithin a schematically illustrated appliance 50, which may be used tosupport the device 10 and which includes heating element 57 forregulating the temperature of the reaction chamber 22 in device 10.

FIG. 3B is a schematic illustration of an analytical device 10 nestedwithin appliance 50, which may be used to support the device 10 andwhich includes the heating element 53 for regulating the temperature ofthe reaction chamber 22 in device 10.

FIG. 4 is a schematic longitudinal cross sectional view of a deviceaccording to the invention that includes a solid substrate 14, on whichis machined mesoscale flow channel 20 connected to inlet ports 16 andPCR reaction chamber sections 22, with a transparent cover 12 adhered tothe surface of the substrate.

FIG. 5 is a perspective view of the device of FIG. 4.

FIG. 6A is a schematic illustration of analytical device 10 nestedwithin appliance 50, which may be used to support the device 10, andwhich includes heating elements 57 for regulating the temperature of thereaction chamber sections 22 in device 10.

FIG. 6B is a schematic illustration of analytical device 10 nestedwithin appliance 50, which may be used to support the device 10 andwhich includes heating element 57 for regulating the temperature of thereaction chamber section 22A in device 10.

FIG. 7 is a schematic plan view of a substrate 14 microfabricated withmesoscale PCR chamber sections 22A and 22B, in fluid communication witha detection chamber comprised of a fractally bifurcating system of flowchannels 40 symmetrically disposed on the substrate.

FIG. 8 is a cross sectional perspective view of a flow channel 20 insubstrate 14 with cell or debris filtering protrusions 80 extending froma wall of the channel.

FIG. 9 is a cross sectional perspective view of a flow channel 20 insubstrate 14 with cell piercing protrusions 90 extending from a wall ofthe channel.

FIG. 10 is a schematic plan view of a mesoscale PCR analytical deviceincluding PCR chamber sections 22A and 22B microfabricated in thesilicon substrate 14.

FIG. 11 is a schematic plan view of another mesoscale PCR analyticaldevice including a PCR chamber 22A microfabricated in the siliconsubstrate 14.

FIG. 12 is a schematic plan view of an analytical device fabricated witha series of mesoscale chambers suitable for implementing a variety offunctions including cell sorting, cell lysing and PCR analysis.

FIG. 13 is a schematic plan view of an analytical device fabricated witha pair of fractally bifurcating flow channels 40.

FIGS. 14, 15 and 16 illustrate top plan views of different embodimentsof a mesoscale filter 24 microfabricated in flow channel 20 in ananalytical device 10.

FIG. 17 is a schematic perspective view of an apparatus 60 used incombination with device 10 for viewing the contents of device 10.

FIG. 18 is a schematic cross sectional view of the apparatus 60 of FIG.17.

Like reference characters in the respective drawn figures indicatecorresponding parts.

DETAILED DESCRIPTION

The invention provides a family of small, mass produced, typicallyone-use devices for conducting a polynucleotide polymerization reactionto enable the rapid amplification of a polynucleotide in a fluid sample.The devices comprise a solid substrate, typically on the order of a fewmillimeters thick and approximately 0.2 to 2.0 centimeters square,microfabricated to define a sample inlet port and a mesoscale flowsystem. The mesoscale flow system includes at least one sample flowchannel extending from the inlet port and at least one polynucleotidepolymerization reaction chamber in fluid communication with the flowchannel. The arrangement of channels, chambers, and multiple portsfacilitates the sequential, properly timed, and volumetrically correctaddition of sample and reagents within the device. The reaction chamberand the flow channels preferably have a mesoscale dimension, i.e. across sectional dimension on the order of 0.1 to 500 μm. The preferreddepth of the reaction chamber is on the order of 0.7 to 100 μm, andpreferred width is on the order of 2.0 to 500 μm. The preferred depth ofthe flow channels is on the order of 0.1 to 100 μm, and the preferredwidth is on the order of 2.0 to 200 μm.

In one embodiment, the devices may be utilized to conduct a polymerasechain reaction (PCR) in the reaction chamber (PCR chamber). The PCRchamber is provided with reagents required for a polymerase chainreaction including the sample polynucleotide, a polymerase such as Taqpolymerase, nucleoside triphosphates, a first primer hybridizable withthe sample polynucleotide, and a second primer hybridizable with asequence complementary to the polynucleotide, wherein the first andsecond primers define the termini of the polymerized productpolynucleotide. The polymerase chain reaction may be performed,according to methods established in the art (Maniatis et al., MolecularCloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press,1989). The device may include means for thermally cycling the contentsof the chamber such that, in each cycle, temperature is controlled todehybridize double stranded polynucleotide to produce single strandedpolynucleotide, and then to anneal the primers and allow polynucleotidepolymerization. In addition, other polynucleotide polymerizationreactions known in the art may be utilized including the isothermal invitro amplication of DNA by a restriction enzyme/DNA polymerase system.Walker et al., Proc. Natl. Acad. Sci U.S.A., 89:392-396 (1992). A ligasechain reaction also may be utilized. Backman, K. Clin. Chem., 38:457-458(1992).

In one embodiment, the device also may include means for detecting theamplified polynucleotide. The devices may be used to implement a varietyof automated, sensitive and rapid polynucleotide analyses including theanalysis of polynucleotides in cells or in solution. At the conclusionof the assay the devices typically are discarded. The use of disposabledevices eliminates contamination among samples. The sample and reactionmixture at all times can remain entombed, and the low volume simplifieswaste disposal.

Analytical devices having mesoscale flow channels and reaction chamberscan be designed and fabricated in large quantities from a solidsubstrate material. They can be sterilized easily. Silicon is preferredbecause of the well-developed technology permitting its precise andefficient fabrication, but other materials may be used, such as polymersincluding polytetrafluoroethylenes. The sample inlet and other ports,the mesoscale flow system, including the sample flow channel(s) and thereaction chamber(s) and other functional elements, thus may befabricated in expensively in large quantities from a silicon substrateby any of a variety of micromachining methods known to those skilled inthe art. The micromachining methods available include film depositionprocesses such as spin coating and chemical vapor deposition, laserfabrication or photolithographic techniques such as UV or X-rayprocesses, or etching methods which may be performed by either wetchemical processes or plasma processes. (See, e.g., Manz et al., Trendsin Analytical Chemistry. 10:144-149 (1991)).

Flow channels of varying widths and depths can be fabricated withmesoscale dimensions. The silicon substrate containing a fabricatedmesoscale flow channel may be covered and sealed with a thin anodicallybonded glass cover. Other clear or opaque cover materials may be used.Alternatively, two silicon substrates can be sandwiched, or a siliconsubstrate can be sandwiched between two glass covers. The use of atransparent cover results in a window which facilitates dynamic viewingof contents in the mesoscale flow system. Other fabrication approachesmay be used.

In one embodiment, a PCR analysis may be conducted in the reactionchamber of the devices. As illustrated schematically in FIGS. 1 and 2,the device 10 may include a silicon substrate 14 microfabricated withinlet ports 16, a mesoscale flow channel 20, and PCR chamber 22. Thepolynucleotide sample and the reagents required for the polymerizationreaction are added, and the products withdrawn (if necessary) throughflow channel 20 and reaction chamber 22 through inlet ports 16 which arefabricated on either end of the flow channel 20. The substrate 14 iscovered with a glass or plastic coverslip 12. During an analysis, thedevice 10 may be used in combination with an appliance, such asappliance 50 shown schematically in FIG. 3A. Appliance 50 includes anesting site 58 for holding the device 10, and for registering ports,e.g., ports 16 on device 10, with a flow line 56 in the appliance. Apump 52 in appliance 50 is used to deliver a sample and/or reagents fromflow line 56 in the appliance to the reaction chamber 22 via the inletports 16.

The appliance 50 may include a heating/cooling element 57 forcontrolling the temperature within the PCR chamber, e.g., an electricalheating element and/or a refrigeration coil. The electrical heatingelement may alternatively be integrated into the substrate 10, withcontacts for power mated to matching electrical contacts in theappliance below the reaction chamber 22. Alternatively, as shown in FIG.3B, the appliance may include a heating means 53, such as a laser, orother source of electromagnetic energy, disposed over the reactionchamber in device 10. Alternatively, the laser may be disposed in theappliance below the reaction chamber. A microprocessor in the appliancemay be used to regulate the heating element in order to provide atemperature cycle in the PCR chamber between a temperature suitable fordehybridization, e.g. 94° C., and a temperature suitable for annealingand polymerization, e.g. 65° C. A thermocouple may also be provided inthe substrate in electrical contact with the appliance, to allow themicroprocessor to detect and maintain the temperature cycles in thereaction chamber. A cooling element, such as a miniature thermoelectricheat pump (Materials Electronic Products Corporation, Trenton, N.J.),may also be included in the appliance for adjusting the temperature ofthe reaction chamber. In another embodiment, in the appliance shown inFIG. 3B, the temperature of the reaction chamber can be regulated by atimed laser pulse directed at the reaction chamber through glass cover12, so as to allow sequential heating and cooling of the sample to therequired temperatures for the PCR cycle. The thermal properties ofsilicon enable a rapid heating and cooling cycle.

The analytical devices also may be utilized in combination with anappliance for viewing the contents of the mesoscale channels in thedevices. The appliance in one embodiment may comprise a microscope forviewing the contents of the mesoscale channels in the devices. Inanother embodiment, a camera may be included in the appliance, asillustrated in the appliance 60 shown schematically in FIGS. 17 and 18.The appliance 60 is provided with a housing 62, a viewing screen 64 anda slot 66 for inserting a chip into the appliance. As shown in crosssection in FIG. 17, the appliance 60 also includes a video camera 68, anoptical system 70, and a tilt mechanism 72 for holding device 10, andallowing the placement and angle of device 10 to be adjusted manually.The optical system 70 may include a lens system for magnifying thechannel contents, as well as a light source. The video camera 68 andscreen 64 allow changes in sample fluid properties, such as flowproperties or color, induced by the presence of polymerizedpolynucleotide, to be monitored visually and optionally recorded usingthe appliance.

In another embodiment, illustrated schematically in FIGS. 4, 5 and 6A, amesoscale PCR chamber may be microfabricated with multiple sections,e.g., two sections 22A and 22B, connected by flow channel 20B. In thisembodiment, section 22A is heated to a temperature suitable fordehybridization and section 22B is heated to a temperature suitable forannealing and polymerization. During an analysis, the device 10 may beplaced in appliance 50 (FIG. 6A). The appliance 50 is provided withmeans 57 for controlling the temperature of the reaction chambersections. Alternatively, a laser may be used to heat the sections. Athermocouple can be included in the substrate to monitor thetemperatures of the sections of the reaction chamber, and its output maybe used to control thermal input with the aid of a microprocessor. Inoperation, a pump 52 in the appliance is used to deliver thepolynucleotide sample and the required PCR reagents from flow line 56through inlet port 16A to section 22A. The pump 52, which also may becontrolled by a microprocessor in the appliance, is then used to cyclethe sample continually between sections 22A and 22B, through channel 20Bto implement a continual polymerase chain reaction cycle, while port 16Bserves as a vent. When the reaction is complete, the pump 52 inappliance 50 may be used to deliver the sample through port 16B and line56 in the appliance to port 59 to recover the product. Of course, threeor more chambers may be used, each of which are maintained at atemperature suitable for conducting a particular reaction.

In another embodiment, in the device 10 shown in FIGS. 4, 5 and 6B, aheating element may be used to heat section 22A to a temperaturesuitable for dehybridization of double stranded DNA, e.g. 94° C., whilesection 22B and channel 20B, which connects sections 22A and 22B, arespaced apart from section 22A such that upon transport of a heatedsample from section 22A to section 22B, heat is dissipated sufficientlyto permit the temperature of the sample to fall to the temperaturerequired for annealing and polymerization before the sample is returnedto section 22A for further cycling. This may be achieved readily assilicon has a relatively high thermal conductivity and the area ofinterface between the liquid sample and the substrate is quite high. Inthis embodiment, microprocessors in the appliance 50 are used to controlpump 52, which regulates the flow cycle of the sample between sections22A and 22B. Thus, a dynamic thermal equilibrium creates a temperaturegradient along the flow path between the chambers, and appropriatetemperatures are achieved in both using a single heating source. Otherdesigns are possible. For example, the annealing and polymerizationreactions could be implemented in different sections of a single PCRchamber, set at different optimized temperatures.

The polymerase chain reaction may be implemented using any thermostablepolynucleotide polymerase, such as Taq polymerase. Reagents such as Taqpolymerase may be added to a sample and then delivered through an inletport to the mesoscale reaction chamber, or the reagents may be deliveredto the reaction chamber independently of the sample through a separateinlet port.

The capacity of the devices is very small and therefore the amount ofsample fluid required for an analysis is low. For example, in a 1 cm×1cm silicon substrate, having on its surface an array of 500 grooveswhich are 10 microns wide×10 microns deep×1 cm (10⁴ microns) long, thevolume of each groove is 10⁻³ μL and the total volume of the 500 groovesis 0.5 μL. The low volume of the mesoscale flow systems allows assays tobe performed on very small amounts of a liquid sample (<5 μl). Themesoscale flow systems of the devices may be microfabricated withmicroliter volumes, or alternatively nanoliter volumes or less, whichadvantageously limits the amount of sample and/or reagent fluidsrequired for an assay.

The devices of the invention provide mesoscale polynucleotidepolymerization reaction chambers which may be used for the rapidamplification of a polynucleotide in a biological fluid sample. Thedevice may also include a means for detecting the amplifiedpolynucleotide product disposed either in the substrate or in theappliance. The presence of amplified polynucleotide product in thedevice can be detected by any of a number of methods includingmonitoring the pressure or electrical conductivity of sample fluidsentering and/or exiting the reaction chamber in the mesoscale flowsystem. The presence of amplified polynucleotide product also can bedetected by a binding assay with a labeled probe such as a labeledoligonucleotide or antibody probe, or by gel electrophoresis.

In one embodiment, the amplified polynucleotide product can be detectedby using a detection chamber fabricated in the mesoscale flow system inthe substrate in fluid communication with the reaction chamber. Thedetection chamber is provided with a binding moiety capable of bindingto the amplified polynucleotide. The binding moiety may comprise, e.g.,a polynucleotide or antibody probe. The detection chamber may befabricated in accordance with methods disclosed in U.S. Ser. No.07/877,702, filed May 1, 1992, the disclosure of which is incorporatedherein by reference. The device may be used in combination with anappliance containing a microprocessor for detecting and recording dataobtained during an assay.

In one embodiment, the mesoscale detection chamber may be provided withan inert substrate, e.g., a bead or other particle, capable of bindingto the polymerized polynucleotide, to cause detectable agglomeration ofthe beads in the presence of polymerized polynucleotide product.Particle induced agglomeration can be enhanced by the attachment of abinding moiety, such as an antibody, to the particle.

Antibodies or other binding moieties capable of binding to thepolymerized polynucleotide may be introduced into the detection chamber,or may be coated, either chemically or by absorption, onto the surfaceof the detection region, or alternatively, onto the surface of an inertparticle in the detection region, to induce binding, giving a positivetest for the polynucleotide. Techniques for the chemical activation ofsilaceous surfaces are well developed, particularly in the context ofchromatography. (See, e.g., Haller in: Solid Phase Biochemistry, W. H.Scouten, Ed., John Wiley, N.Y., pp 535-597 (1983); and Mandenius et al.,Anal. Biochem. 170:68-72 (1988)). In one embodiment, the binding moietymay comprise an antibody, and immunoassay techniques known in the artcan be performed in the detection region. (See, e.g., Bolton et al.,Handbook of Experimental Immunology, Weir D. M., Ed., BlackwellScientific Publications, Oxford, 1986, Vol. 1, Chapter 26, for a generaldiscussion of immunoassays).

An optically detectable label such as a fluorescent molecule orfluorescent bead may be attached to the binding moiety to enhancedetection of the polymerized polynucleotide. Alternatively a secondlabeled substance, such as a fluorescent labeled antibody may bedelivered through the flow system to bind to the boundpolynucleotide/binding moiety complex in the detection region to producea "sandwich" including an optically detectable moiety indicative of thepresence of the analyte. The binding of the amplified polynucleotide tothe binding moiety in the detection region may be detected, e.g.,optically, either visually or by machine, through a transparent windowdisposed over the detection region. In one embodiment, the production ofamplified polynucleotide may be detected by the addition of a dye suchas ethidium bromide, which exhibits enhanced fluorescence upon bindingto double stranded polynucleotide. Higuchi et al., Biotechnology, 10:413(1992).

The detection chamber may also be provided with a labelled complementarypolynucleotide capable of binding to one of the strands of the amplifiedpolynucleotide, e.g., a labeled polynucleotide immobilized on a bead, toenable the detection of polymerized polynucleotide product by means ofbead agglutination. Polynucleotide hybridization techniques known in theart may be utilized. Maniatis et al., Molecular Cloning: A LaboratoryManual, 2nd ed., Cold Spring Harbor Press, 1989); Vener et al., Anal.Chem., 198:308-311 (1991). Polynucleotide probes may be attached to,e.g., a submicron latex particle. Wolf et al., Nucleic Acids Research,15:2911-2926 (1987).

Polynucleotide polymerization also can be detected using a detectionregion sensitive to flow restriction caused by the presence ofpolymerized polynucleotide produced.in the reaction chamber, as isdisclosed in U.S. Ser. No. 07/877,701, filed May 1, 1992, the disclosureof which is incorporated herein by reference. The presence of amplifiedpolynucleotide also may be detected by sensing the pressure orelectrical conductivity of the fluid samples entering and exiting theflow system. The conductivity may be measured, e.g., using electricalcontacts which extend through the substrate and which mate withelectrical contacts in an appliance used in combination with the device.Electrical contacts can be fabricated by known techniques of thermalgradient zone melting. (See Zemel et al., in: Fundamentals andApplications of Chemical Sensors, D. Schuetzle and R. Hammerle, Eds.,ACS Symposium Series 309, Washington, DC, 1986, p. 2.)

Amplified polynucleotide in the reaction chamber can be detected bymonitoring the pressure of the sample fluids. For example, in a device10, nested in appliance 50, illustrated schematically in FIG. 6A, thepressure detectors 54 connected to sample fluid entering and exiting themesoscale flow system through ports 16 will allow the detection ofpressure decreases caused by the presence of polymerized product andresulting clogging or flow restriction. A mesoscale pressure sensor alsomay be fabricated directly on the silicon substrate. Angell et al.,Scientific American 248:44-55 (1983).

Polynucleotide polymerization can be detected by the use of a mesoscaleflow system sensitive to flow restriction, constructed with a "fractal"pattern, i.e., a pattern of serially bifurcating flow channels. Thefractally bifurcating channels may be fabricated on a silicon substratewith reduced dimensions at each bifurcation, providing sequentiallynarrower flow channels. FIG. 7 shows a schematic plan view of asubstrate 14 fabricated with a fractally bifurcating system of flowchannels 40 connected via channel 20 to ports 16 and a PCR reactionchamber comprising sections 22A and 22B. The presence of amplifiedpolynucleotide product in a sample will influence the flowcharacteristics within the fractal. The channels 40 in this embodimentare symmetrically disposed and have a sequentially narrower diametertowards the center of the fractal. Flow through this fractal issensitive to changes in fluid viscosity caused by the presence ofpolymerized product. Alternatively a more complex fractal flow systemmay be utilized, as illustrated in FIG. 13. FIG. 13 illustrates a pairof fractally bifurcating flow channels 40A and 40B. The fractal flowchannel 40A is constructed with sequentially narrower flow channelstowards the center of the fractal, resulting in an enhanced sensitivityto flow restriction.

Flow restriction in the fractal region can be detected, e.g., optically,through a transparent cover over the detection region. Alternatively,one or more pressure sensors may be utilized to detect pressure changesdue to changes in fluid properties caused by the presence of amplifiedpolynucleotide in or beyond the fractal flow paths. Changes inconductivity upon polynucleotide production also may be readily detectedthrough electrical conductivity sensors in contact with the flow region.For example, clogging of the fractal region 40 which blocks flow frominlet port 16A to outlet port 16B could be detected by a conventionalconductivity probe 17 whose output is indicative of the presence orabsence of aqueous fluid in the outflow channel. Binding moieties suchas labeled antibodies or polynucleotide probes may be included in thefractal region, e.g. immobilized, or on a solid phase reactant such as abead, to bind to the product polynucleotide to induce flow restrictionin the fractal flow path.

In one embodiment, the mesoscale flow system includes a chamber forlysing cells from a sample in preparation for downstream polynucleotideanalysis. The devices also may include a region adapted to separate aparticular type of cell in a heterogeneous cell population. The cellseparation region includes immobilized binding moieties immobilized onstructures within the substrate which selectively reversibly bind atarget cell via a characteristic cell surface molecule such as aprotein. Other cells in the sample pass downstream and are discardedinto a sump or through an exit port. Flow may be continued to wash thecells, e.g., with a flow of buffer. At higher flow rates and pressures,the washed cells are sheared from the surfaces, released from theseparation region, and move downstream to a lysis means, which lyse thecells prior to PCR analysis of an intracellular RNA or DNA molecule.

The cell lysing means typically is disposed in the flow path between thecell separation region (if any) and the polynucleotide polymerizationreaction chamber to allow the cells to be lysed prior to analysis for anintracellular polynucleotide. As illustrated in FIG. 9, the cell lysingmeans may comprise cell membrane piercing protrusions 90 extending froma surface of a flow channel 20. As fluid flow is forced through thepiercing protrusion 90, cells are ruptured. In another embodiment, thecell lysis means may simply comprise a region of restrictedcross-sectional dimension which implements cell lysis upon applicationof sufficient flow pressure. The cell lysis means may also comprisesharp edged pieces of silicon trapped within a mesoscale lysis chamber.An appliance which includes means, such as a pump, for forcing the cellcontaining sample into the cell lysis means, causes cell lysis uponapplication of sufficient flow pressure, and subsequently delivers thesample through the flow system to the reaction chamber. In anotherembodiment, the cell lysis means may comprise a cell lysing agent. Celllysing agents known in the art may be utilized.

Reagents may be added to the reaction chamber from a separate inlet portin the substrate in fluid communication with the reaction chamber. Afilter, microfabricated in the flow channel on the silicon substrate,can be used to filter cell debris prior to polynucleotide analysis. Inone embodiment, shown in FIGS. 14, 15 and 16, the filter 24 in device 10may comprise a mesoscale flow channel of reduced diameter in comparisonwith channel 20. In operation, sample flows from sample flow channel 20Athrough filter 24. Sample filtrate then exits filter 24 and flowsthrough channel 20B. The filter 24 is microfabricated with depths andwidths on the order of 0-1 to 20 μm, while flow channels 20A and 20Bhave maximum depths and widths on the order of approximately 500 μm. Asillustrated in FIG. 8, the surface of a flow channel 20 may also includeprotrusions 80 constituting a cellular sieve for separating cells bysize upstream from the PCR analysis chamber. As cell samples are flowedthrough the flow channel, typically under low pressure, only cells smallenough to pass between the protrusions 80 reach downstream functionalelements. These cells subsequently can be delivered through a cell lysisregion, then to a PCR reaction chamber for analysis.

In another embodiment, paramagnetic or ferromagnetic beads may beprovided within the mesoscale flow system, which can be moved along theflow system by an external magnetic field, e.g., in the appliance. Thebeads may be used to transport reagents between functional elements inthe device, or to displace a sample, a reagent or a reaction mixture. Inone embodiment, a polynucleotide probe may be immobilized on themagnetic beads enabling the beads to bind to amplified polynucleotide.Magnetic beads comprising a coating of polynucleotide probe may betransported through the flow system to the reaction chamber at the endof an assay to bind to the polymerized polynucleotide product. The boundpolymerized polynucleotide then may be transported on the magnetic beadsto a detection or purification chamber in the flow system, or to acollection port.

One embodiment of the invention, illustrated in FIG. 10, is a device 10comprising a substrate 14 microfabricated with a mesoscale PCR chambercomprising sections 22A and 22B, which are connected by flow path 20B.The PCR chip 10 is used in combination with an appliance, such asappliance 50, shown in FIG. 6A, which contains a nesting site forholding the chip. The appliance 50 is provided with flow paths 56 matedto ports 16A, 16B, 16C and 16D in device 10. The appliance also includesvalves that allow the ports 16A, 16B, 16C and 16D to be mechanicallyopened and closed. In one embodiment, the flow systems of the devicesmay be maintained at a hydraulically full volume, and valves in theappliance, or alternatively, in the devices, may be utilized to directfluid flow. Sections 22A and 22B of the PCR chamber are heated to 94° C.and 65° C., respectively, to provide a melting temperature and anannealing temperature as required for PCR. As discussed above, reactionchamber sections may be heated by means of an electrical contactintegrated in the substrate below the sections, which can mate withelectrical contacts in the appliance. Alternatively, an optical lasermay be used to heat the reaction chamber sections through a glass coverdisposed over the substrate. A heat sensor may be provided in thesubstrate, in electrical contact with the appliance. A microprocessor inthe appliance can be used to control the temperature of the reactionchamber sections and the flow of fluid in the flow system.

In operation, initially, with the channels and chambers full of buffer,port 16A and 16C are open while 16B and 16D are closed. A pump 52 in theappliance delivers the sample fluid and, optionally, reagents requiredfor PCR such as Taq polymerase, primers and nucleoside triphosphates,via port 16A, through filter 24, to reaction chamber section 22A. Port16A next is closed and 16B is opened, and the pump 52 in the applianceis used to reciprocate fluid flow in cycles through flow channel 20Bbetween section 22A, where polynucleotide dehybridization occurs, andsection 22B, where annealing and polymerization occurs. Port 16C can beused to vent the system, and also optionally to deliver Taq polymerase,nucleoside triphosphates, primers, and other reagents. When thepolymerase cycling reaction is complete, e.g., after 30-35 cycles, port16C is closed, port 16D is opened, and the pump in the appliance isactuated to deliver the reaction products from PCR chamber sections 22Aand 22B to detection chamber 22C, which contains, e.g., a polynucleotidecomplementary to the amplified sense and/or antisense strand,immobilized on beads 92. Polymerization product is detected by observingthe agglutination of beads 92, e.g., visually through a translucentcover disposed over the detection region.

Another embodiment is illustrated in FIG. 11. The function, structure,and operation of this device is identical to that shown in FIG. 10,except that it comprises a single PCR reaction chamber 22A. The deviceis used in combination with an appliance such as appliance 50 shown inFIG. 3A. The device includes means for heating and cooling reactionchamber 22A alternatively to a temperature required for melting and atemperature required for annealing and polymerization.

In operation, the appliance is used to deliver a sample containingpolymerase and other reagents required for PCR through inlet port 16A toreaction chamber 22A. Ports 16A and 16D are then closed using a valveconnected in the appliance, while port 16B and 16C remain open. Theheating element in the appliance is then utilized to thermally cycle thereaction chamber between a temperature suitable for dehybridization anda temperature suitable for annealing and polymerization. When the PCRreaction cycle is complete, port 16C is closed, port 16D is opened andthe sample is delivered to detection chamber 22B which contains apolynucleotide probe, e.g., immobilized upon beads 92. A positive assayfor the polynucleotide is indicated by agglutination of thepolynucleotide probe in the detection chamber.

The invention will be understood further from the following, nonlimitingexamples.

EXAMPLE 1

A polymerase chain reaction is performed in the device illustratedschematically in FIG. 11. To perform a PCR analysis to detect apolynucleotide in a cell, a sample cell lysate is added to a bufferedsolution of Taq polymerase, nucleoside triphosphates, polynucleotideprimers and other reagents required for PCR. The cell sample lysate isdelivered via the appliance through entry port 16A to PCR reactionchamber 22A. Ports 16A and 16D are closed by means of valves included inthe appliance, while port 16B and 16C are open. The microprocessor andtemperature control element in the appliance are used to implement atemperature cycle in reaction chamber 22A between 94° C., forpolynucleotide dehybridization, and 65° C., for polymerase reaction.After the polymerase chain reaction is complete, port 16C is closed, 16Dopened, and the pump in the appliance connected to port 16B used todeliver the sample from the PCR reaction chamber 22A through flowchannel 20B to the detection chamber 22B. Detection chamber 22B containsbeads 92 comprising a surface immobilized complementary polynucleotidecapable of binding the amplified polynucleotide. The agglutination ofthe beads caused by hybridization reaction between the amplifiedpolynucleotide and the complementary polynucleotide is observed througha window disposed over the detection region 22B, and provides a test forthe presence of amplified polynucleotide product.

EXAMPLE 2

FIG. 12 depicts schematically a device 10 including substrate 14 used toseparate a nucleic acid from a subpopulation of cells in a mixture in abiological fluid sample, and then to perform an assay for a particularnucleotide sequence. Microfabricated on device 10 is a mesoscale flowpath 20 which includes a cell separation chamber 22A, a cell lysischamber 22B, a filter region 24, a PCR reaction chamber comprisingsections 22C and 22D, and a fractal detection region 40. The mesoscaleflow system 20 is also provided with fluid entry/exit ports 16A, 16B,16C and 16D. The device is used in combination with an appliance, suchas appliance 50, shown in FIG. 6A.

Initially, the valves in the appliance are used to close ports 16C and16D, while ports 16A and 16B are open. A sample containing a mixture ofcells is directed to the sample inlet port 16A by the pump 52 in theappliance, and flows through the mesoscale flow path 20 to separationchamber 22A. Chamber 22A contains binding moieties immobilized on thewall of the chamber which selectively bind to a surface molecule on adesired type of cell in the sample. Remaining cellular components exitthe substrate via port 16B. After binding of the desired cell populationin chamber 22A, flow with buffer is continued, to wash and assureisolation of the cell population. Next port 16B is closed and 16C isopened. Flow is then increased sufficiently to dislodge the immobilizedcells. Flow is continued, forcing cells through membrane piercingprotrusions 90 in chamber 22B, which tear open the cells releasingintracellular material. Sample flow continues past filter 24, whichfilters off large cellular membrane components and other debris, tomesoscale PCR chamber section 22C, which is connected to PCR chambersection 22D by flow channel 20B. Taq polymerase, primers and otherreagents required for the PCR assay next are added to section 22Dthrough port 16C from a mated port and flow path in the appliance,permitting mixing of the intracellular soluble components from theseparated subpopulation of cells and the PCR reagents. With port 16Aclosed, a pump in the appliance connected via port 16B is used to cyclethe PCR sample and reagents through flow channel 20B between sections22C and 22D, set at 94° C. and 65° C. respectively, to implement pluralpolynucleotide melting and polymerization cycles, allowing theamplification of product polynucleotide. The valves in the appliancenext are used to close port 16C and to open port 16D. The pump in theappliance connected to port 16B is then used to direct the amplifiedpolynucleotide isolated from the cell population to a detection regioncomprised of a fractally bifurcating series of flow paths 40. Flowrestriction in the fractal region 40 serves as a positive indicator ofthe presence of amplified polynucleotide product and is detectedoptically through a glass cover disposed over the detection region.

It will be understood that the above descriptions are made by way ofillustration, and that the invention may take other forms within thespirit of the structures and methods described herein. Variations andmodifications will occur to those skilled in the art, and all suchvariations and modifications are considered to be part of the invention,as defined in the claims.

What is claimed is:
 1. A device for amplifying a preselectedpolynucleotide in a sample by conducting a polynucleotide amplificationreaction, the device comprising:a solid substrate which ismicrofabricated to define:a sample inlet port; a mesoscale flow systemcomprising:a sample flow channel extending from said inlet port; and apolynucleotide amplification reaction chamber, in fluid communicationwith said flow channel; said sample flow channel and said reactionchamber having at least one cross-sectional dimension of width or depthwhich is about 0.1 to 500 μm, said chamber and said channel being ofdissimilar dimension; and a fluid exit port in fluid communication withsaid flow system; and means for thermally cycling the contents of saidchamber whereby in each cycle the temperature is controlled todehybridize double stranded polynucleotide and to permit the in vitroamplification of a preselected polynucleotide.
 2. The device of claim 1,wherein said amplification reaction is a polymerase chain reaction(PCR), and wherein said amplification chamber contains: a preselectedpolynucleotide, a polymerase, nucleoside triphosphates, a first primerhybridizable with said sample polynucleotide, and a second primerhybridizable with a nucleic acid comprising a sequence complementary tosaid polynucleotide, wherein said first and second primers define thetermini of the polynucleotide product of the polymerization reaction;andwherein said means for thermally cycling comprises means forthermally cycling the contents of said chamber between a temperaturecontrolled to dehybridize double stranded polynucleotide thereby toproduce single stranded polynucleotide, to permit annealing of saidprimers to complementary regions of single stranded polynucleotide, andto permit synthesis of polynucleotide between said primers, thereby toamplify said preselected polynucleotide.
 3. The device of claim 1wherein said amplification chamber comprises:a first section at atemperature which dehybridizes double stranded polynucleotide; a secondsection at a temperature which permits annealing of complementaryregions of single stranded polynucleotide and permits amplification ofpolynucleotide; a flow path disposed between said first and secondsections; and wherein said device includes:means for repeatedlytransporting the contents of said chamber between at least said firstand said second sections to implement plural cycles of amplification ofsaid polynucleotide.
 4. The device of claim 3wherein said second sectionand said flow path are spaced apart from said first section such thatupon transport of the contents of said chamber from said first sectionto said second section, the sample cools passively to a temperaturesufficient to permit annealing of single stranded polynucleotide and topermit amplification of said preselected polynucleotide.
 5. The deviceof claim 3 further comprising means for separately thermally controllingsaid first and said second sections.
 6. The device of claim 4 furthercomprising means for thermally controlling said first section.
 7. Thedevice of claim 5 or 6 wherein said means for thermally controllingcomprises electrical resistance means in said substrate.
 8. The deviceof claim 5 or 6 wherein said means for thermally controlling comprisesmeans for providing electromagnetic energy to said amplificationchamber.
 9. The device of claim 1 wherein said solid substrate comprisesmicrofabricated silicon.
 10. The device of claim 1 further comprising anappliance for use in combination with said substrate, said appliancecomprising:means for holding said substrate; and fluid input meansinterfitting with an inlet port on said substrate.
 11. The device ofclaim 10 further comprising pump means for passing fluid through theflow system of said substrate when it is held in said holding means. 12.The device of claim 11 wherein said appliance further comprises areagent reservoir and means for delivering a reagent to said flowsystem.
 13. The device of claim 10 wherein said means for thermallycycling comprises an electromagnetic energy source; andwherein saidelectromagnetic energy source is provided in said appliance.
 14. Adevice for amplifying a preselected polynucleotide in a sample, thedevice comprising:a solid substrate microfabricated to define:a sampleinlet port; a mesoscale flow system comprising:a sample flow channelextending from said inlet port; and a polynucleotide amplificationreaction chamber, in fluid communication with said flow channel,containing a preselected polynucleotide and polynucleotide amplificationreagents; said sample flow channel and said reaction chamber having atleast one cross-sectional dimension of width or depth which is about 0.1to 500 μm, said chamber and said channel being of dissimilar dimension;and a fluid exit port in fluid communication with said flow system; andmeans for thermally cycling the contents of said chamber whereby, ineach cycle, temperature is controlled to dehybridize double strandedpolynucleotide, and to permit synthesis of polynucleotide, thereby toamplify said preselected polynucleotide.
 15. The device of claim 14wherein said flow system further comprises a detection chamber in fluidcommunication with said amplification chamber.
 16. The device of claim14 wherein said amplification chamber comprises:a first section at atemperature which dehybridizes double stranded polynucleotide; a secondsection at a temperature which permits annealing of single strandedpolynucleotide, and which permits amplification of polynucleotide; and aflow path disposed between said first and second sections; and whereinthe device further comprises means for repeatedly transporting thecontents of said chamber between said first and said second sections toimplement plural cycles of amplification of said polynucleotide.
 17. Thedevice of claim 14 further comprising an appliance for use incombination with said substrate, said appliance comprising:a nestingsite for holding said substrate, which comprises fluid input meansinterfitting with an inlet port on said substrate.
 18. The device ofclaim 17 wherein said device includes electrical contacts fabricated inthe substrate; andwherein said nesting site further includes anelectrical connection for interfitting with said electrical contact insaid substrate.
 19. The device of claim 17 wherein the appliance furthercomprises pump means for passing fluid through the flow system of saidsubstrate when it is held in said holding means.
 20. The device of claim17 wherein the appliance further comprises a power supply.
 21. A methodfor amplifying a preselected polynucleotide in a sample by conducting apolynucleotide amplification reaction, the method comprising:(i)providing a device comprising:a solid substrate microfabricated todefine:a sample inlet port; a mesoscale flow system comprising:a sampleflow channel extending from said inlet port; and a polynucleotideamplification reaction chamber in fluid communication with said flowchannel; said sample flow channel and said reaction chamber having atleast one cross-sectional dimension of width or depth which is about 0.1to 500 μm, said chamber and said channel being of dissimilar dimension;and a fluid exit port in fluid communication with said flow system; andmeans for thermally regulating the contents of said chamber at atemperature controlled to permit amplification of said preselectedpolynucleotide; (ii) delivering, through said inlet port and saidmesoscale flow system to said reaction chamber, a sample polynucleotideand reagents required for an in vitro polynucleotide amplificationreaction; and (iii) thermally controlling the contents of said chamberto permit amplification of said polynucleotide.
 22. The method of claim31 wherein said amplification reaction is a polymerase chain reaction(PCR);wherein, in step (i), said means for thermally controllingcomprises means for thermally cycling the contents of said chamber;wherein step (ii) includes the step of adding to said amplificationchamber: a polymerase, nucleoside triphosphates, a first primerhybridizable with said sample polynucleotide, and a second primerhybridizable with a nucleic acid comprising a sequence complementary tosaid polynucleotide, and wherein said first and second primers definethe termini of the polynucleotide product of the polymerizationreaction; and wherein step (iii) includes the step of thermally cyclingthe contents of said chamber whereby, in each cycle, the temperature iscontrolled to dehybridize double stranded polynucleotide thereby toproduce single stranded polynucleotide, to permit annealing ofcomplementary regions of single stranded polynucleotide, and to permitsynthesis and polymerization of polynucleotide between said primers. 23.The method of claim 22 wherein said amplification chamber comprises:afirst section at a temperature which dehybridizes double strandedpolynucleotide; a second section at a temperature which permitsannealing of complementary regions of single stranded polynucleotide andpermits amplification of polynucleotide; a flow path disposed betweensaid first and second sections; wherein the device furtherincludes:means for repeatedly transporting the contents of said chamberbetween said first and said second sections; and wherein step (iii)includes the step of repeatedly transporting the contents of saidchamber between said first and said second sections to implement pluralcycles of amplification of polynucleotide.
 24. The method of claim 23wherein said first section is controlled at a temperature to dehybridizedouble stranded polynucleotide; andwherein said second section and saidflow path are spaced apart from said first section such that upontransport of the contents of said chamber from said first section tosaid second section, the sample cools substantially to a temperature toanneal and polymerize; and wherein step (iii) includes the step ofrepeatedly transporting the contents of said chamber between said firstand second sections to polymerize said polynucleotide.
 25. A device foramplifying a preselected polynucleotide in a sample by conducting apolynucleotide amplification reaction, the device comprising:a solidsubstrate microfabricated to define:a sample inlet port; a mesoscaleflow system comprising:a sample flow channel extending from said inletport; and a polynucleotide amplification chamber, in fluid communicationwith said flow channel, said chamber containing reagents for amplifyinga preselected polynucleotide in vitro; said sample flow channel and saidreaction chamber having at least one cross-sectional dimension of widthor depth which is about 0.1 to 500 μm, said chamber and said channelbeing of dissimilar dimension; and a fluid exit port in fluidcommunication with said flow system.
 26. The device of claim 25 whereinsaid reagents comprise reagents for conducting a polymerase chainreaction.
 27. The device of claim 25 wherein said reagents comprisereagents for conducting a ligase chain reaction.